Mutect2 Usage, how can I make it work?

Question

[m-hayashi]

Dear Users,

I tried to detect SNV and indel using Mutect2, as below;

java -Xmx4g -jar /path/to/gatk-4.0.0.0/gatk-package-4.0.0.0-local.jar Mutect2
--input normal_rmdup-realgin.bam tumor_rmdup-realign.bam --output mutect.vcf
--tumor-sample tumor_rmdup-realign.bam -R ~/home/path/to/hg19genome.fa

then, the error has occured "A USER ERROR has occurred: Bad input: BAM header sample names [whatever]does not contain given tumor sample name tumor_rmdup-realign.bam" (Note:'normal' and 'tumor' are other names, and directory name is changed, practically.)

I'd like to know the reason of this error, the header of BAM is invalid?

the header of BAM shows below

@HD     VN:1.4  GO:none SO:coordinate
@SQ     SN:chrM LN:16571
@SQ     SN:chr1 LN:249250621
@SQ     SN:chr2 LN:243199373
@SQ     SN:chr3 LN:198022430
@SQ     SN:chr4 LN:191154276
@SQ     SN:chr5 LN:180915260
@SQ     SN:chr6 LN:171115067
@SQ     SN:chr7 LN:159138663
@SQ     SN:chr8 LN:146364022
@SQ     SN:chr9 LN:141213431
@SQ     SN:chr10        LN:135534747
@SQ     SN:chr11        LN:135006516
@SQ     SN:chr12        LN:133851895
@SQ     SN:chr13        LN:115169878
@SQ     SN:chr14        LN:107349540
@SQ     SN:chr15        LN:102531392
@SQ     SN:chr16        LN:90354753
@SQ     SN:chr17        LN:81195210
@SQ     SN:chr18        LN:78077248
@SQ     SN:chr19        LN:59128983
@SQ     SN:chr20        LN:63025520
@SQ     SN:chr21        LN:48129895
@SQ     SN:chr22        LN:51304566
@SQ     SN:chrX LN:155270560
@SQ     SN:chrY LN:59373566
@RG     ID:1    PU:whatever     LB:whatever     SM:whatever     PL:illumina
@PG     ID:bwa  VN:0.7.12-r1039 CL:/bin/bwa mem /path/to/hg19genome.fa /home/path/to/tumor_R1.fastq.gz /home/path/to/tumor_R2.fastq.gz    PN:bwa
@PG     ID:GATK IndelRealigner  CL:knownAlleles=[] targetIntervals=tumor_rmdup.intervals LODThresholdForCleaning=5.0 consensusDeterminationModel=USE_READS entropyThreshold=0.15 maxReadsInMemory=150000 maxIsizeForMovement=3000 maxPositionalMoveAllowed=200 maxConsensuses=30 maxReadsForConsensuses=120 maxReadsForRealignment=20000 noOriginalAlignmentTags=false nWayOut=null generate_nWayOut_md5s=false check_early=false noPGTag=false keepPGTags=false indelsFileForDebugging=null statisticsFileForDebugging=null SNPsFileForDebugging=null

I'd appreciate giving kindness, thanks.

Sincerely.

[NB]

Have you tried the GATK forum ?

[m-hayashi]

Thank you for reply, but not yet.

Answer 1

[shubhra.bhattacharya]

Try adding Read group. This can be done using picard addorreplacereadgroups. Maybe mutect isn't able to read which sample is what in the bam files.

http://broadinstitute.github.io/picard/ :About picard tools.

A Readgroup added bam file looks like:

@SQ SN:KQ714869.1 LN:201 @SQ SN:KQ714870.1 LN:201 @SQ SN:KQ714871.1 LN:201 @SQ SN:KQ714872.1 LN:201 @SQ SN:KQ714873.1 LN:201 @SQ SN:KQ714874.1 LN:201 @SQ SN:KQ714875.1 LN:201 @SQ SN:KQ714876.1 LN:201 @SQ SN:KQ714877.1 LN:201 @SQ SN:KQ714878.1 LN:201 @SQ SN:KQ714879.1 LN:201 @SQ SN:KQ714880.1 LN:201 @SQ SN:KQ714881.1 LN:140232 @RG ID:CRME_AB_S_ID PU:CRME_AB_S_PU LB:CRME_AB_S_LB SM:CRME_AB_S_SM PL:CRME_AB_S_PL

@RG indicates my sample info. as you can see from above.

[m-hayashi]

thank you for your advice.

However, realign, included in my bam file name, means that these bams are realigned by IndelRealigner of GATK after adding read group by picard. Then, it is indicated that header is invalid?

my process is below;

fastq > mapping by bwa mem (with no option) > sort by samtools > rmdup by samtools >

add read groups by picard > Indelrealigner by GATK

For comparison, varscan2 has worked well with these bams. At this time, we need to know variation of detected-mutation and VAF of them in each methods (including other methods, EBcall, Strelka, etc) for validating threshold of VAF by NGS in our protocol.

[shubhra.bhattacharya]

Try ValidateBam(Picard tools) file and try to see what maybe going wrong with the bam file as it will give you a overall diagnosis about the bam file. my RGID looks like this:

@RG ID:CRME_AB_S_ID (this ID is the same as my sample name)

@RG ID:1 PU:whatever LB:whatever SM:whatever PL:illumina Your RG ID is 1. Is this in correspondence?